Effect of epigenetic treatment on SST2 expression in neuroendocrine tumour patients

Several preclinical studies have uncovered that epigenetic drugs can upregulate somatostatin receptor subtype 2 (SST 2 ) expression in neuroendocrine tumour (NET) models, 1,2 which could be of eminent importance for NET patients with low tumoural SST expression. In a prospective clinical proof-of-concept trial involving nine advanced NET patients with low SST expression, we were able to show that epigenetic treatment with the histone deacetylase (HDAC) inhibitor valproic acid and the DNA methyltransferase (DNMT) inhibitor hydralazine did not lead to an increase in tumour-uptake of 68 Ga-DOTATATE, contradicting the in vitro data.

TA B L E 2 Change in study parameters of neuroendocrine tumour patients at baseline and after 1 and 2 weeks of epigenetic treatment

Laboratory parameters
Haemoglobin, mmol/L (IQR) 8.5 (8.1, 9.2) 8.5 (7.7, 9.2) 8.1 (7.6, 8.6) .05 Thrombocytes, ×10 9  adjusted to target a serum concentration of 75-120 μg/ml. 6 Hydralazine dosage remained unchanged unless adjusted for tolerability. Treatment effect was evaluated after 2 weeks by the change in 68 Ga-DOTATATE uptake on PET/CT. The last two patients (lung NET, rectum NET) completed the trial without hydralazine due to emerging insights from the in vitro studies, which were performed simultaneously in three human NET cell lines BON-1 (pancreatic NET), GOT1 (small intestinal NET) and NCI-H727 (lung NET). Here, effects of valproic acid sodium salt and hydralazine on SST 2 mRNA and protein levels as well as 111 In-DOTATATE uptake were assessed (details in the Supplementary Appendix). At the end of the 2-week epigenetic treatment period, none of the NET patients had an increase in 68 Ga-DOTATATE uptake grade (Table 2). No change in median 68 Ga-DOTATATE uptake in any NET sites was observed, and there was even a tendency for reduced uptake in pri-mary tumours (Figures 1 and S1). These findings were independent of tumour aetiology, metastatic location or drug treatment. Meanwhile, a significant median (IQR) increase of 27% (4.1, 46.4) in uptake was observed in the kidneys, p = .02, independent of the study medication. A limitation of our study is the restricted patient number, but given the lack of effects in any of the patients with different NET origins, this protocol is unlikely to affect tumoural SST 2 expression in vivo. All patients reported known side effects of the study medication (details in the Supplementary Appendix), and no serious adverse events occurred during the study.
In all cell lines tested, treatment with valproic acid led to a significant increase of SST 2 mRNA levels and 111 In-DOTATATE uptake, p < .001, respectively ( Figure 2). An increase in the SST 2 staining intensity per cell was observed in BON-1 and NCI-H727 cells (p < .01), but not in GOT1 cells, possibly due to the high baseline SST 2 F I G U R E 1 Change in peak uptake of 68 Ga-DOTATATE on PET/CT at baseline and after 2-week epigenetic treatment in patients with neuroendocrine tumours with low somatostatin receptor expression. The upper panel showing changes in tumour lesions, the lower panel showing changes in physiological uptake. Patients were prepared according to our local protocol, which includes the drinking of 1 L of water in 2 h before injection. Imaging was performed from scull base to thighs after median (interquartile range [IQR]) 60 min (59-65) injection with an activity of 118-MBq (103-121) 68 Ga-DOTATATE. For each patient, at least two tumour target lesions, including the primary if applicable, were defined on the initial 68 Ga-DOTATOC PET/CT. Peak standard uptake value (SUV) was calculated for every lesion as well as for the liver, kidneys and spleen. *p < .05 according to Wilcoxon signed-rank test expression levels ( Figure S2). Meanwhile, an increase in SST 2 mRNA levels was seen only for the stronger hydralazine dose in BON-1 cells (p < .001) and GOT1 cells (p < .05), but hydralazine decreased mean (SD) SST 2 mRNA expression levels in NCI-H727 cells by 15% (13), p < .05. No changes in SST 2 protein expression and 111 In-DOTATATE uptake were seen following incubation with hydralazine in all cell lines. The combined treatment of valproic acid with the stronger hydralazine dosage led to an additional mean (SD) increase in SST 2 mRNA expression levels in BON-1 cells of 120% (72), p < .001, whereas no additional effect was seen for GOT1 cells, and even an inhibitory mean effect (SD) of 73% (34), p < .001, was observed in NCI-H727. No synergistic or antagonistic effect on 111 In-DOTATATE uptake and SST 2 staining intensity per cell was observed for the combined treatment.
Our study shows, for the first time, that contrary to the promising in vitro and in vivo data on epigenetic upreg-ulation of SST 2 expression, epigenetic treatment did not translate into the stimulation of 68 Ga-DOTATATE uptake in NET patients with low baseline SST expression.
This appears to be in contrast to the study with five SST-positive patients who received vorinostat-treatment for 4 days, 5 but their observed change in the maximum standard uptake value (SUV max ) of 1.3 could lack clinical relevance. Combined these studies might imply that either epigenetic upregulation of SST 2 expression is only effective in patients with sufficient baseline 68 Ga-DOTATATE uptake, or the epigenetic effect depends on the epidrugs used or the drug levels achieved in patients are not sufficient to induce upregulation. The importance of choice and dosage of the epidrugs was shown by the effect of the DNMT inhibitor hydralazine, exhibiting only mild effects in pharmacologically unreachable dosages despite good efficiency observed in other tumours. 7 The Netherlands) supplemented with primers and probes was used. SST 2 expression was determined relative to three housekeeping genes (GUSB, HPRT1 and ACTB) using the QuantStudio 7 Flex RT-qPCR system with QuantStudio Real-Time PCR software v1.5. Immunohistochemistry was performed using rabbit monoclonal anti-SST 2 IgG (NB-49-015, 1:25 dilution, NeoBiotech, Nanterre, France). Stained cells were visualized with the NanoZoomer 2.0 HT (Hamamatsu Photonics, Hamamatsu City, Japan) and SST 2 staining intensity per cell was assessed using the CellProfiler software (version 4.0.7, www.cellprofiler.org). Internalization studies were performed with 111 In-DOTATATE. 111 InCl 3 (Curium Pharma, Petten, The Netherlands) was used to radiolabel DOTATATE (Bachem AG, Bubendorf, Switzerland) with a molar activity of 50 MBq/nmol. Data are shown as mean with the standard deviation of three (mRNA expression levels and radiolabelled 111 In-DOTATATE uptake) or two (immunohistochemistry) independent experiments. Data were normalized to control values, all set at 100%. HE, hematoxylin eosin. *p < .05, **p < .01, ***p < .001 according to one-way ANOVA analysis with Tukey post hoc test after log transformation of data also be the observed non-specific effect of increased renal uptake in our patients. Although changes in uptake measures of up to 25% SUV max between two scans have been described, 9 the increase in renal uptake was seen in 78% of our patients in the second PET/CT. As all patients underwent the same hydration protocol before the scan and no changes in kidney function were noted, this could signify that the epigenetic treatment is not tumour specific and also activates basal expression of SST 2 in renal tissue. 10 In conclusion, short-term epigenetic treatment with valproic acid and hydralazine had no stimulating effect on 68 Ga-DOTATATE uptake in nine patients with well-differentiated NETs of various origins with low baseline SST expression, contradicting preclinical findings. Clinical trials with alternative epigenetic drugs or in patients with positive baseline SST 2 expression may be able to clarify whether epigenetic treatment has a role in the treatment of NETs; however, a potential increase in renal uptake should be closely monitored.

A C K N O W L E D G E M E N T
We would like to thank the Radiopharmaceutical Chemistry group (Radiology and Nuclear Medicine, Erasmus MC, The Netherlands) for radiolabelling 111 In-DOTATATE used for the uptake experiments performed in this study.

F U N D I N G I N F O R M AT I O N
This study was supported by the Erasmus MC Foundation. JR was supported by a grant from the Swiss National Science Foundation (P2BSP3-181720).

C O N F L I C T O F I N T E R E S T
RF received research support from Ipsen, Strongbridge and Corcept as well as speaker fees from HRA Pharma, Novartis, Ipsen. WWDH received research support from AAA-Novartis, speaker fees from Ipsen and AAA-Novartis and is on the advisory board of AAA. TB received speaker fees, research support from AAA and is on the advisory board of AAA. JH received speaker fees from Ipsen and is on the advisory board of Novartis. 1